DNA polymerase, primer pair, dNTP, Mg++, and other buffers are required for PCR components which are essential for molecular diagnostic test or nucleic acid diagnostic test. The amplification of the interested target gene having high specificity and accuracy is required to use such a reaction widely.
Robust PCR performance requires highly sensitive analytical PCR such as single-copy DNA molecule detection (Wabuyele, M. B et al. Single Mol., 2001, 2: 13-21), bloodborne infection (Elnifro, E. M. et al. Clin. Microbiol. Rev., 2000, 13: 559-570), or the like.
Generally, in order to carry out PCR, the information about the target gene to be detected is obtained, and primer & probe design is performed. At this time, off-target amplification often occurs although the primer sequence and length designed are designed to well hybridize only to template DNA/RNA at annealing temperatures. It is considered that the reason for this is because of low-temperature conditions during PCR master-mix preparation and thermal cycler ramping to the initial denaturation temperature (Chou, Q et al. Nucleic Acids Res., 1992, 20: 1717-1723). Under such a condition, the primers are present at a higher concentration than the target, and the primers react non-specifically with partially complementary sites, or the primers react with each other. These non-specific primer complexes competitively react to the desired target site reaction, thereby impairing sensitivity to serve as a cause of high background. In particular, primer dimers may form complex mixtures with primer artifacts during PCR.
Several methods are widely used for increasing the specificity of PCR, and one method among them employs a hot start technique. This technique increases the temperature to maintain the specificity of the primer/target hybridization reaction so that the DNA polymerase interferes with the premature extension when preparing the pre-PCR mixture (Alexandre V. L et al., Lebedev A. V. et al., Nucleic Acids Res., 2008, 36: e131.). Other methods are used in which the reaction components are physically separated so as not to cause a PCR reaction (Quin chou et al., Nucleic Acids Res., 1992, 20: 1717-1723), accessary proteins are used (Clark, D. R. et al. US Pat. No. 2006057617), an antibody against a DNA polymerase is used, and a Mg++-pyrophosphate complex is formed to prevent a non-specific reaction at a low temperature (Bioneer).
In addition to increasing the specificity by controlling the reaction components, several methods have been reported to modify the primer to increase the specificity. Among them, there are a method of using to include competitor sequence, a method of using a secondary structure of a primer, a method of increasing a hybridization selectivity, and a dual priming oligo method using two types of primers to be attached to each other (for example, Seegene). Further, as a 3′-modification method, a method of blocking primer extension until the 3′→5′ exonuclease has been removed, a method of blocking it by removal through UV irradiation, and a thermal deprotection method have been reported.
These methods have the effect of reducing the non-specific reaction, but they have disadvantages of using additional enzymes, adding extra activation conditions, specific nucleoside modification, difficulty in coverage for variants of the corresponding target gene due to structurally complex primer design, and a decrease of sensitivity.